Hyaluronan-CD44 Promotes Phospholipase C–Mediated Ca2+ Signaling and Cisplatin Resistance in Head and Neck Cancer

Objective  To investigate whether hyaluronan (HA)–CD44 promotes head and neck squamous cell carcinoma (HNSCC) cisplatin resistance and whether HA-CD44 promotes phospholipase C (PLC)–mediated Ca²⁺ signaling to alter cisplatin sensitivity in HNSCC.

Design  Cell line study.

Main Outcome Measures  Tumor cell growth with the chemotherapeutic drug cisplatin was measured in the presence or absence of HA, anti-CD44 antibody plus HA, and other inhibitors of the PLC-mediated Ca²⁺ signaling pathway. Ca²⁺ mobilization was measured with fluorescence spectrophotometry using the Ca²⁺ binding dye Fura/2AM.

Results  In the absence of HA, cisplatin inhibited tumor cell growth. The addition of HA, but not HA plus anti-CD44 antibody, resulted in a 5-fold reduced ability of cisplatin to cause HNSCC cell death, suggesting that HA can promote CD44-dependent cisplatin resistance. Fluorescence spectrophotometry demonstrated that HA can promote CD44-dependent Ca²⁺ mobilization in HNSCC. On the other hand, the presence of U73122, a PLC inhibitor, and 2-aminoethoxydiphenyl borate, an inositol-1,4,5-triphosphate receptor inhibitor, eliminated HA-mediated Ca²⁺ mobilization and HA-mediated cisplatin resistance in these cell lines.

Conclusions  Our results indicate that HA-CD44 signaling influences cisplatin sensitivity in HNSCC cell growth. In particular, HA-CD44 promotion of PLC-mediated Ca²⁺ signaling plays a role in cisplatin resistance in HNSCC cells. Perturbation of this HA-CD44–mediated signaling pathway may be a promising target to overcome cisplatin resistance in HNSCC.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide.¹ Advanced-stage HNSCC continues to have poor 5-year survival rates (0%-40%), which have not significantly improved in the last 30 years. Understanding the mechanisms underlying HNSCC tumor progression and resistance to standard treatment is critical to improving outcomes for this disease. Tumor progression (ie, tumor invasion, migration, and metastasis) is determined by both genetic mutations and the tumor’s microenvironment, including the interaction between tumor cells and extracellular matrix molecules. Tumor drug resistance involves multiple mechanisms, including reduced drug accumulation, decreased apoptosis, alterations in drug target, and increased DNA damage repair. The interaction between tumor cells and their microenvironment can lead to the activation of oncogenic signaling pathways, which promote both cancer progression and drug resistance.

Hyaluronan (HA) is a glycosaminoglycan component of the extracellular matrix and has well-known biophysical properties. More recently, HA has been studied with regard to its interaction with various cell signaling pathways.^2-4 Hyaluronan is capable of binding to several transmembrane receptors, including CD44, which is expressed in many different normal and malignant cell types.^5-13 In cancer cells, HA interaction with CD44 promotes multiple cell signaling pathways, which influence tumor cell progression behaviors, including abnormal adhesion, migration, and invasion.^7-10,13 The interaction of HA-CD44 with these signaling pathways is incompletely understood, and little is known about the role of HA and CD44 in HNSCC.

Phospholipase C (PLC)–mediated Ca²⁺ signaling appears to interact with HA-CD44. In several cell model systems, HA-CD44 signaling has been found to promote intracellular Ca²⁺ mobilization.^11,12 Hyaluronan stimulation of CD44 leads to kinase phosphorylation of PLC to its active phosphorylated form. Phosphorylated PLC catalyzes the conversion of phosphatidylinositol-4,5-bisphosphate to inositol-1,4,5-triphosphate (IP₃) and diacylglycerol. IP₃ acts as a second messenger, binding to the IP₃ receptor. to induce release of Ca²⁺ from intracellular stores (Figure 1).^11-14 The HA-CD44 interaction also promotes intracellular Ca²⁺ mobilization by recruitment of the IP₃ receptor into membrane lipid rafts.¹²

In this study, we sought to determine whether HA promotes CD44-dependent HNSCC drug resistance and whether HA-CD44 interacts with PLC-mediated Ca²⁺ signaling to promote tumor cell survival. For this investigation, we used the chemotherapeutic drug cisplatin. Although the biochemical mechanism of action of cisplatin remains incompletely understood, the current accepted paradigm is that the drug binds to nuclear DNA, leading to interference with normal transcription and/or DNA replication mechanisms.¹⁵ Cisplatin administration appears to result in the activation of several signal transduction pathways, including those that involve p53 and mitogen-activated protein kinase.¹⁶ If the cisplatin-DNA adducts are not efficiently processed by cell machinery, the eventual result is cell death. Cisplatin is one of the most common and potent anti–head and neck cancer chemotherapeutic drugs, and it has a response rate as a single agent in head and neck cancer of approximately 30%.¹⁷

Cisplatin sensitivity of 2 HNSCC cell lines in the presence of HA was determined. We also studied the role of HA in promoting Ca²⁺ mobilization and found that specific inhibitors of the PLC-mediated Ca²⁺ signaling pathway could eliminate HA-mediated Ca²⁺ mobilization and HA-mediated cisplatin resistance in HNSCC.

The cell line SCC-4 (American Type Culture Collection, Manassas, Va) was derived from a primary oral tongue squamous cell carcinoma removed from a 55-year-old man. The SCC-4 cells were maintained in a 1:1 mix of F-12 and Dulbecco modified Eagle medium supplemented with essential and nonessential amino acids, vitamins, and 10% fetal bovine serum. The cell line HSC-3 (Japan Cancer Research Resources Bank, Tokyo) was established in 1985 from a primary oral tongue squamous cell carcinoma removed from a 64-year-old man. The HSC-3 cells were maintained in Dulbecco modified Eagle medium supplemented with essential and nonessential amino acids, vitamins, and 10% fetal bovine serum.

The SCC-4 or HSC-3 cells grown in serum-free media were solubilized in 50mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (pH, 7.5), 150mM sodium chloride, 20mM magnesium chloride, 1.0% NP-40, 0.2mM sodium orthovanadate, 0.2mM phenylmethylsulfonyl fluoride, 10 μg/mL of leupeptin, and 5 μg/mL of aprotinin. After brief centrifugation, the samples were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis using 4% to 12% polyacrylamide gels. Separated polypeptides were then transferred onto nitrocellulose filters. After blocking nonspecific sites with 2% bovine serum albumin, the nitrocellulose filters were incubated with rat anti-CD44 antibody followed by incubating with horseradish peroxidase–labeled anti-rat IgG. The blots were then developed with an enhanced chemiluminescence system (ECLTM system; Amersham Co, Piscataway, NJ).

Monoclonal rat antihuman CD44 antibody (clone, 020; isotype, IgG2b; obtained from CMB-TECH Inc, San Francisco, Calif) used in this study recognizes a common determinant of the CD44 class of glycoproteins, including CD44s. The PLC inhibitor U73122 and the IP₃ receptor inhibitor 2-aminoethoxydiphenyl borate (2-APB) were obtained from CalBiochem (EMD Biosciences, San Diego, Calif).

Logarithmically growing cell lines were cultured, washed, counted, and plated at 3000 cells per well in triplicate wells of 96-well plates and incubated in serum-free media overnight. The following day, the cells were treated with various concentrations of cisplatin without or with HA (50 μg/mL), anti-CD44 antibody, the IP₃ receptor inhibitor 2-APB (50μM), or the PLC inhibitor U73122 (5μM). Two days later, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were performed according to the manufacturer’s protocol (Roche Diagnostics, Mannheim, Germany). The MTT assay measures cell survival based on mitochondrial conversion of MTT from a soluble tetrazolium salt into an insoluble colored formazan precipitate, which is dissolved in dimethyl sulfoxide and quantitated by spectrophotometry. The percentage of absorbance relative to controls was plotted as a linear function of drug concentration. The 50% inhibitory concentration (IC₅₀) was identified as a concentration of the drug required to achieve a 50% growth inhibition relative to untreated controls.

The HNSCC cells grown in serum-free media (untreated or pretreated with anti-CD44 antibody, U73122 [5μM], or 2-APB [50μM]) were first incubated with 10μM Fura-2/AM (CalBiochem) for 1 hour at room temperature in a buffer solution that contained 145mM sodium chloride, 5mM potassium chloride, 0.1mM magnesium chloride, 5mM glucose, and 15mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (pH, 7.3). Cells were subsequently washed 3 times with the same buffer, then placed in a 20-μL chamber alternately illuminated with 200-millisecond flashes of 340 and 380 nm every 10 milliseconds (monitoring emission wavelength of 510 nm) using a dual-wavelength fluorescence imaging system (Intracellular Imaging Inc, Cincinnati, Ohio). A phosphate-buffered solution that contained HA (50 μg/mL) was added to the chamber, and the imaging measurements were continued. The concentration of intracellular Ca²⁺ was determined by the following equation: Ca²⁺ = K_d × [(R − R_min)/(R_max − R)] × F/B, where Ca²⁺ is intracellular Ca²⁺, K_d is the dissociation constant of Fura-2/AM for Ca²⁺, R is the ratio of the Fura-2/AM fluorescence excited at 340 nm divided by the fluorescence excited at 380 nm, R_min and R_max are minimal and maximal fluorescence ratios, respectively, obtained in ionomycin in the presence of 7mM ethyleneglycotetraacetic acid or 2.0mM Ca²⁺, and F and B are the fluorescence voltage signals at 380 nm in 50μM ionomycin in the presence of 7mM ethyleneglycotetraacetic acid and 2mM Ca²⁺, respectively.

CD44 is constitutively expressed in 2 HNSCC cell lines, HSC-3 and SCC-4. Cell lysates from oral cavity primary cancers, SCC-4 and HSC-3, were separated by gel electrophoresis and immunoblots obtained. The anti-CD44 antibody that we used recognizes a common determinant of the CD44 class of glycoproteins, including standard (CD44s) and other CD44 variant isoforms. Cell lysates from 2 HNSCC cell lines, SCC-4 and HSC-3, demonstrate CD44 isoform expression (Figure 2). A strong signal at 85 to 90 kDa (CD44s) was seen. In addition, other larger bands were seen corresponding to high-molecular-weight splice variants of CD44 (CD44v), consistent with various other reports of CD44 isoform expression in head and neck cancers.^5,6,8 Therefore, we chose to use the HSC-3 and SCC-4 HNSCC cell lines to study CD44 oncogenic signaling.

Hyaluronan promotes CD44-dependent cisplatin resistance in HSC-3 and SCC-4. The introduction of HA (50 μg/mL) to serum-free HNSCC cell cultures (SCC-4 and HSC-3) led to an increase in cellular proliferation (data not shown). These results suggest that these 2 HNSCC cell lines are sensitive to HA-dependent signaling. Tumor cell growth with the anti–head and neck cancer drug cisplatin was measured via MTT assay in the presence or absence of HA (50 μg/mL) or anti-CD44 antibody plus HA (Figure 3 and Table). In the absence of HA, cisplatin inhibited tumor cell growth (with IC₅₀ values of 20μM and 4μM, respectively, for SCC-4 and HSC-3). The addition of HA reduced the ability of cisplatin to cause HNSCC cell death (IC₅₀ values of 100μM and 20μM, respectively, for SCC-4 and HSC-3), indicating that HA can promote these tumor cells to become cisplatin resistant. Furthermore, pretreatment of HNSCC tumor cells with anti-CD44 antibody followed by the addition of HA eliminated the HA-mediated drug resistance, suggesting that HA acts through CD44 to promote cell survival in the presence of cisplatin.

Hyaluronan promotes CD44-dependent Ca²⁺ mobilization in HSC-3 and SCC-4. To investigate whether HA-CD44 influences the PLC-mediated Ca²⁺ signaling pathway in HNSCC, we performed fluorescent spectrophotometry with HSC-3 or SCC-4 cells loaded with Fura-2/AM, a fluorescent Ca²⁺ binding dye. The Fura-2/AM–labeled cells were subsequently treated with HA (50 μg/mL), pretreated with anti-CD44 antibody followed by HA treatment, or pretreated with various inhibitors of PLC-mediated Ca²⁺ signaling followed by HA treatment. Figure 4 demonstrates that HA stimulated intracellular Ca²⁺ mobilization in HSC-3, and this HA-mediated rise in intracellular Ca²⁺ was eliminated by pretreatment with anti-CD44 antibody (blocks HA binding to CD44), U73122 (PLC inhibitor), and 2-APB (IP₃ receptor inhibitor). These results support the notion that HA-CD44 induces Ca²⁺ mobilization in HNSCC, and this mobilization requires both PLC and IP₃ receptor.

The PLC inhibitor (U73122) eliminates HA-mediated cisplatin resistance and enhances cisplatin sensitivity in HSC-3 and SCC-4. To investigate whether cisplatin resistance in HNSCC might be mediated through HA-CD44 interaction with PLC-mediated Ca²⁺ signaling, we performed MTT assays with HSC-3 and SCC-4 in the presence of increasing concentrations of cisplatin combined with the PLC inhibitor U73122 (Figure 5 and Table). At a concentration of 5μM, U73122 alone caused little inhibition of tumor cell proliferation with or without HA. However, the same concentration of U73122, when combined with cisplatin, eliminated the HA-mediated protection and led to increased cisplatin sensitivity in both HNSCC cell lines.

The IP₃ receptor inhibitor (2-APB) eliminates HA-mediated cisplatin resistance in HSC-3 and SCC-4. Since PLC is known to hydrolyze phosphatidylinositol-4,5-bisphosphate to diacylglycerol and IP₃, we next used the IP₃ receptor inhibitor 2-APB to see whether this inhibitor would also eliminate HA-mediated cisplatin resistance (Figure 6 and Table). At a concentration of 50μM, 2-APB alone caused little inhibition of tumor cell proliferation with or without HA. However, when the same concentration of 2-APB was combined with cisplatin, the HA protection from cisplatin was eliminated. These results suggest that both PLC and IP₃ receptor play important roles in HA-CD44–mediated cisplatin resistance.

Standard chemotherapeutic drugs demonstrate limited effectiveness against HNSCC, highlighting the need for a better understanding of the molecular mechanisms by which head and neck tumors resist drug-induced destruction. The HA-CD44 interaction with various signaling pathways, such as the PLC-mediated Ca²⁺ signaling pathway, is being increasingly examined as a possible mechanism for both tumor progression and chemotherapy resistance. We wondered whether HA-CD44 could promote drug resistance in HNSCC. We also wondered whether HA-CD44 could promote PLC-dependent Ca²⁺ mobilization in HNSCC and whether the up-regulation of this pathway could be involved in tumor cell survival and drug resistance.

In this study, we found that HA can promote cisplatin resistance in 2 HNSCC cell lines. Our data also suggest that this HA-mediated cisplatin resistance involves PLC-mediated Ca²⁺ signaling. We found that HA promotes CD44-dependent Ca²⁺ mobilization in HNSCC that can be blocked by inhibitors of PLC and the IP₃ receptor. Furthermore, these same inhibitors are capable of eliminating HA-mediated cisplatin resistance, and the PLC inhibitor U73122 can also enhance cisplatin sensitivity.

Figure 1 shows our current proposed model of an HA-CD44–PLC-mediated Ca²⁺ signaling pathway. In this model, the interaction of HA-CD44 with PLC leads to Ca²⁺ mobilization with subsequent tumor cell survival and resistance to cisplatin. It is possible that HA may also mediate cisplatin resistance through other signaling pathways; CD44 is known to be linked to various transmembrane and intracytoplasmic signaling pathways, such as those involving epidermal growth factor receptor, mitogen-activated protein kinase, phosphoinositide 3-kinase, and others.^3,4,7-10,13 The interaction of CD44 with these receptor-mediated signaling pathways has been shown to promote various tumor progression behaviors, including tumor cell growth, migration, invasion, and metastasis. We are currently investigating whether the interaction of HA-CD44 with these signaling receptor pathways also promotes drug resistance.

The inhibitor U73122 inhibits all isoforms of PLC (eg, PLC-α, PLC-β, and PLC-γ-1). Since the various PLC isoforms are regulated differently, further investigation is required to determine what PLC isoforms are important in HA-mediated Ca²⁺ mobilization and cisplatin resistance. Future studies that target specific PLC isoforms, such as using small interfering RNA techniques, would help clarify the role of the different PLC isoforms. It is known that 2-aminoethoxydiphenyl borate is a membrane-permeable agent that inhibits IP₃ receptor–induced Ca²⁺ release.¹⁸ Studies in various mammalian cell systems have shown that 2-APB blocks the IP₃ receptor–mediated release of Ca²⁺ through store-operated channels from intracellular storage sites. The results in our study using this inhibitor demonstrate that the IP₃ receptor mediates HA-induced Ca²⁺ mobilization and cisplatin resistance.

The Ca²⁺ released from intracellular storage sites is known to mediate a variety of important cell processes, including alterations in the cytoskeleton, migration, invasion, and promotion of cell survival.^11-14,19,20 Hyaluronan-mediated Ca²⁺ signaling in endothelial cells is involved in nitric oxide production and cell adhesion, migration, and proliferation.^11-13 In T helper 1 cells, HA-CD44 interacts with PLC-γ-1 to induce transendothelial migration.¹⁹ In addition, PLC-mediated Ca²⁺ signaling has been shown to promote cytoprotection in human intestinal cells and tumor cell invasion in HNSCC.^14,20 The results of the current investigation demonstrate that PLC-mediated Ca²⁺ signaling also promotes cisplatin resistance in HNSCC.

Cisplatin is one of the most important anti–head and neck cancer chemotherapies used today. However, cisplatin resistance is a frequently encountered problem. Various mechanisms of cisplatin resistance in head and neck cancer have been proposed, and P53 mutation was shown to correlate with cisplatin sensitivity in a panel of 23 HNSCC cell lines.²¹ Glutathione S-transferase amplification was associated with cisplatin resistance in 10 HNSCC cell lines and 10 primary tumor samples.²² Another study²³ found a correlation between glutathione expression, but not nuclear factor κB expression, and in vitro cisplatin sensitivity. A study²⁴ of 29 patients treated with combination cisplatin and radiotherapy demonstrated decreased progression-free and overall survival with c-erbB2 expression. These studies suggest that multiple unique pathways may promote cisplatin resistance in HNSCC.

Hyaluronan-CD44 signaling has been linked to chemotherapy resistance in several tumor models. Hyaluronan oligosaccharides, which competitively inhibit HA-CD44 interaction, were shown to inhibit anchorage-independent growth in a lung and breast cancer cell line, and this effect appeared to be mediated through suppression of the phosphoinositide 3-kinase/protein kinase B pathway.^25,26 Furthermore, these HA oligomers also sensitized a doxorubicin-resistant breast carcinoma cell line to a variety of chemotherapeutic agents.²⁶ Another study²⁷ found that enhanced expression of a variant CD44 isoform was associated with carmustine resistance in a colon carcinoma cell line. These studies suggest that HA-CD44 oncogenic signaling could play a key role in multidrug resistance in HNSCC. We are currently investigating HNSCC sensitivity to several other chemotherapeutic drugs, including methotrexate, etoposide phosphate, and paclitaxel. Our preliminary data suggest a similar HA-protective effect with these agents.

In summary, HA promoted cisplatin resistance, whereas perturbation of HA-CD44–mediated PLC/Ca²⁺ signaling led to enhanced cisplatin sensitivity in HNSCC cells. The results of our study suggest that targeting HA-CD44–mediated signaling pathways, such as the PLC/Ca²⁺ pathway, may be a potential therapeutic strategy for HNSCC tumors resistant to standard chemotherapy.

Correspondence: Steven J. Wang, MD, 4150 Clement St, 112B, San Francisco, CA 94121 (swang@ohns.ucsf.edu).

Submitted for Publication: February 16, 2005; final revision received May 11, 2005; accepted May 31, 2005.

Financial Disclosure: None.

Acknowledgment: This work was supported by a Northern California Institute for Research and Education Young Investigator Award; US Public Health Services grants PO1 AR39448, RO1 CA66163, and RO1 CA78633 from the National Institutes of Health, Bethesda, Md; and a Veterans Affairs Merit Review grant. Dr Bourguignon is a Veterans Affairs Career Scientist.